Supplementary Material

Disruption of spatiotemporal hypoxic signaling causes congenital heart disease

Xuejun Yuan, Hui Qi, Xiang Li, Fan Wu, Jian Fang, Eva Bober, Gergana Dobreva, Yonggang Zhou, Thomas Braun

Max-Planck-Institute for Heart and Lung Research, Department of Cardiac Development and Remodeling, Bad Nauheim Germany





Supplementary Figure 1: Induction of hypoxia responses represses Isl1 expression in vivo. (A) Whole-mount immunostaining of C57/Bl6 E9.0 embryos (14 somites) for Isl1 after chemical induction of hypoxia responses (15 or 30mg CoCl2/kg body weight). Arrows indicate the Isl1+ cardiogenic region. Representative images from 2 independent experiments are shown. Scale bar: 100 μm. (B) Representative images of E9.0 (14 somites) C57/Bl6 embryos immunostained for ISL1 and NKX2.5 after CoCl2 treatment are shown. PBS- injected mice were used as control. Arrows indicate cardiac mesoderm and arrowheads indicate the heart tube. Analyzed embryos were randomly selected from two different litters for each condition. Scale bar: 100 μm. (C) Scheme of the strategy to induce hypoxia responses during early embryogenesis. Wild-type C57/Bl6 pregnant mice (E7.5) were housed in a hypoxia chamber containing 10% O2 and 90% N2 for 48 hrs to induce hypoxia responses in vivo. (D) RT-qPCR analysis of Isl1 expression in C57/Bl6 E8.0 embryos (5 somites) after hypoxia exposure. The m34b4 gene was used as a reference for normalization (t-test: *p<0.05, n=3). (E) Analysis of Isl1 expression in C57/Bl6 E9.0 embryos by WISH after hypoxia exposure. Arrows indicate foregut endoderm and arrowheads indicate cardiac mesoderm. Representative images from 2 independent experiments are shown. Scale bar: 100 μm. (F) Scheme of the strategy to induce hypoxia responses in Nkx2.5+/Isl1+-GFP cells isolated from embryoid bodies at day 6. (G) RT-qPCR analysis of Isl1, Nkx2.5 and Flk1 expression in embryoid bodies (EBs) at day 6 after hypoxia exposure for 16 hrs (1% O2). Please note the down-regulation of Isl1 (n=7) but up-regulation of Nkx2.5 (n=3) and Flk1 (n=4). The m34b4 gene was used as a reference for normalization. (t-test: *p<0.05; **p<0.01; n>3). (H) + Analysis of Nkx2.5 cells proliferation in mock or CoCl2 treated E9.5 Nkx2.5-emGFP embryos by immunostaining for phospho-Histone H3 (Ser10) (pH3). The percentages of pH3/GFP double positive cells are shown. At least 3 sections were counted for each embryo + (t-test: ns P>0.05; n=3). (I) TUNEL assay of Nkx2.5 cells after CoCl2 treatment. The percentage of TUNEL-positive Nkx2.5+ cells is shown. At least 4 sections were counted for each embryo (t-test: ns P>0.05; n=3).



Supplementary Figure 2: Isolation and in vitro differentiation of Isl1+ CPCs. (A) Scheme of the generation of Isl1nGFP/+ reporter mice by dual recombinase-mediated cassette exchange. (B) Fluorescence image of GFP positive EBs at day 7 after in vitro differentiation of Isl1nGFP/+ ES cells. Scale bar: 100 μm. (C) Bright field and fluorescence images of control and Isl1nGFP/+ embryos at E8.5 (10 somites). Isl1-GFP-positive cells are indicated by an arrow. Scale bar: 200 μm. (D) Scheme of Isl1+ CPC in vitro differentiation after FACS-based isolation. (E) Immunofluorescence staining of CPCs for ISL1 and NKX2.5 after 2 days culture in differentiation media. Cultivation in differentiation medium increases the number of Nkx2.5+ CPCs. Arrows indicate Isl1-/Nkx2.5+ CPCs and arrowheads indicate Isl1+/Nkx2.5- CPCs. Representative images from 3 independent experiments are shown. Scale bar: 100 μm. (F) Quantification of proliferating Ki67+ (immunostained) CPCs isolated from Isl1nGFP embryos (E8.0 embryos, 5-8 somites) after exposure to normoxia (n=3) or hypoxia (n=4) in vitro. (t-test: **p<0.01). (G) Quantification of the percentage of Nkx2.5+ (immunostained) cells within the population of Isl1+ cells after exposure to hypoxia in vitro. (t-test: *p<0.05, n=4).



Supplementary Figure 3: Generation of transgenic mice expressing Nkx2.5 in Isl1+ cells. (A) Outline of the strategy to express Nkx2.5 in Isl1+ cells. (B) Bright field and fluorescence images of Nkx2.5-IRES-GFP expression in E10.5 embryos (Isl1-creposRosa26Nkx). Scale bar: 500 μm. (C) Western blot analysis to monitor expression of NKX2.5 in Isl1-creposRosa26Nkx embryos at E9.5. Wildtype littermates were used as controls. Histone H3 was used as protein loading control.



Supplementary Figure 4: Hypoxia enhances binding of Sirt1 to the Isl1 promoter. (A) ChIP analysis of HIF1α binding to the Isl1 proximal promoter in differentiating ES cells (EB at day 6) with and without exposure to hypoxia. (B) ChIP analysis of HIF1α binding to the Nkx2.5 distal promoter in differentiating ES cells (EB at day 6). (C) Co-IP assay of HIF1α with SIRT1 or HES1 after CoCl2 treatment of V6.5 ES cells. 2.5% input was used. (D) ChIP analysis of SIRT1 binding to the Isl1 proximal promoter in differentiating ES cells at different time points. (E) ChIP analyses of SIRT1 binding to the Isl1 enhancer and proximal promoter in embryonic hearts at E11.5. (F) ChIP analysis of HIF1α and SIRT1 binding to Isl1 and Nkx2.5 promoters in differentiating ES cells (EB at day 6). Hypoxia enhances binding of HIF1α to the Isl1 and Nkx2.5 promoters and increases binding of SIRT1 to the Isl1 but not the Nkx2.5 promoter. (G) ChIP analysis of SIRT1 and HES1 binding to the Isl1 proximal promoter in differentiating ES cells without and with exposure to 1% O2 for 16 hrs. Note increased binding of SIRT1 and HES1 after exposure to hypoxia. The relative enrichment of SIRT1 is normalized against the input DNA. (t-test: *p<0.05; **p<0.01; n=3). (H) Western blot analysis of HIF1α levels in differentiating ES cells exposed to CoCl2 after infection with a lentivirus expressing an shRNA against Hif1α. α−Tubulin was used as protein loading control. Lanes were run on the same gel but not next to each other.



Supplementary Figure 5: A SIRT1-HES1-containing complex mediates down-regulation of Isl1 expression in CPCs. (A) Western blot analysis of HES1 levels in C2C12 cells after infection with a lentivirus expressing a shRNA against Hes1. Actin was used as protein loading control. (B) Luciferase reporter assays of Isl1 WT and N-box mutated promoters in C2C12 cells under normoxia or hypoxia. Mutation of the N-box increases Isl1 promoter activity and prevents hypoxia-mediated suppression (ANOVA with Tukey’s post hoc test: *p<0.05; ns p>0.05; n=3). (C) Western blot analysis of SIRT1 protein in V6.5 ES cells after infection with a lentivirus expressing Sirt1 shRNA. Actin was used as loading control. (D) RT-qPCR analysis of Isl1, Nkx2.5, Flk1 (EB at E6) and Sma, Myh7 (EB at E8) expression in differentiating V6.5 ES cells after Sirt1 knockdown. The m34b4 gene was used as a reference for normalization (t-test: *p<0.05; **p<0.001; ***p<0.0001; ns p>0.05, n=3). (E) Luciferase reporter assay of the Isl1 promoter in C2C12 cells after knockdown of Sirt1, with and without exposure to hypoxia. Hypoxia fails to reduce Isl1 promoter activity in C2C12 cells after Sirt1 knockdown (t-test: *p<0.05; n=3).



Supplementary Figure 6: Hypoxia responses enhance the activity of SIRT1. (A) RT- qPCR analysis of Sirt1 expression after chemical induction of hypoxia responses (CoCl2 treatment) in E8.5 embryonic hearts. The m34b4 gene was used as a reference for normalization. ANOVA with Dunnett’s post hoc test was used to calculate significance (ns p>0.05; n=4). (B) RT-qPCR analysis of Sirt1 expression in sorted Isl1+ cells after cultivation under normoxia (21% O2) and hypoxia (1% O2) for 16 hours. The β-actin gene was used as a reference for normalization (t-test: ns p>0.05; n=6). (C) NAD+/NADH ratios in sorted Isl1+ cells after cultivation under normoxia (21% O2) and hypoxia (1% O2) for 16 hours. (t-test: ns + p>0.05; n=3). (D) Cellular ROS levels of sorted Isl1 cells under hypoxia (1% O2) and normoxia (21% O2). Two independent experiments were performed generating similar results. (E) Western blot analysis of embryonic hearts isolated from mock (PBS) or CoCl2 treated (15 mg/kg bodyweight) pregnant mice (E8.5, 8-12 somites) and of sorted Isl1+ CPCs exposed to either normoxia (21% O2) or hypoxia (1% O2).



Supplementary Figure 7: Germ line inactivation of Sirt1 leads to multiple cardiac defects. (A) Outline of the strategy to inactivate the Sirt1 gene. (B) Distribution of genotypes at weaning after breeding of heterozygous Sirt1 mutant mice. Asterisk indicates that these mice died 2 weeks after birth. (C) H&E staining of embryonic hearts from Sirt1+/+ (n=6) and Sirt1-/- (n=6) germ line mutants. 6 littermates from 3 different litters were analyzed. Arrows indicate ventricular septum defects (2 out of 6 embryos); arrowhead indicates atrial septum defect (2 out of 6 embryos); Asterisk indicates thinner right ventricular compact layer (4 out of 6 embryos). LA: left atrium; RA: right atrium; LV: left ventricle; RV: right ventricle. Scale bars: 100 μm. (D) RT-qPCR analysis of Isl1 expression in E8.0 (5 somites) Sirt1+/+ WT and Sirt1-/- germ line mutants. The m34b4 gene was used as a reference for normalization (t-test: *p<0.05; n=4 from 2 different litters).



Supplementary Figure 8: Isl1-Cre mediated inactivation of Sirt1 reduces the number of Isl1+/Nkx2.5+ cells in the cardiac mesoderm. (A) Cell tracing of Isl1+ CPCs progeny (green) in E15.5 Isl1-Crepos/Sirt1fl/+/RosaYFP+ and Isl1-Crepos/Sirt1fl/-/RosaYFP+ hearts. Scale bars: 100 μm. (B) RT-PCR analysis of WT and mutant Sirt1 mRNA in Isl1- Crepos/Sirt1fl/+/RosaYFP+ and Isl1-Crepos/Sirt1fl/-/RosaYFP+ E15.5 hearts after sorting of Isl1+ (YFP+) and Isl1- (YFP-) cells. (C) Representative immunofluorescence images of cryosections of E9.5 Isl1-Crepos/Sirt1fl/+ and Isl1-Crepos/Sirt1fl/- hearts stained for ISL1 and NKX2.5 are shown. 3 pairs of Sirt1 heterozygous and mutant littermates from 2 litters were analyzed. Note the decreased number of Isl1+/Nkx2.5+ cells in the cardiac mesoderm after Isl1-Cre mediated knockout of Sirt1.



Table S1

 
No obvious Thinner Muscular OA/VSD DORV PTA Abnormal RV defect myocardium VSD (%) (%) (%) dilation or Treatment (%) (%) (%) hypoplasia (%) Low CoCl2 5 (23%) 6 (27%) 7 (32%) 1 (4,5%) 0 0 9 (41%) (N=22)

High CoCl2 1 (4.5%) 7 (32%) 0 16 3 1 6 (27%) (N=22) (72%) (14%) (4.5%)

Suppl. Table 1: Incidence of cardiac structural defects in CoCl2 treated C57/Bl6 embryos. Summary of cardiac structural defects in CoCl2 treated embryos detected by H&E staining of paraffin sections from E15.5 mouse hearts. The penetrance of cardiac defects in the CoCl2 treated hearts with variable incidence of different CHDs is indicated. DORV, double outlet right ventricle; VSD: ventricular septal defect; PTA: persistent truncus arteriosus (PTA); OA: overriding aorta.

Table S2

Phenotypes No Thinner Muscular OA/VSD Abnormal RV obvious myocardium VSD dilation or Genotypes defect hypoplasia

Sirt1+/+ (n=5) 5 0 0 0 0 Sirt1-/- (n=6) 0 4 (1 out of 4 1 (with 1 0 -CoCl2 with ASD) ASD) Sirt1 WT (n=6) 6 0 0 0 0 (Sirt1fl/+ISl1-Creneg)

Sirt1 conditional 5 2 0 0 0 null mutants (n=7) (Sirt1fl/flISl1-Crepos)

Sirt1 WT (n=8) 2 2 (with 2 4 2 (with (Sirt1fl/+ISl1-Creneg) OA/VSD) OA/VSD) +CoCl2 Sirt1 heterozygotes 11 0 0 0 0 mutants (n=11) Sirt1 conditional 8 0 0 0 0 null mutants (n=8)

Suppl. Table 2: Incidence of cardiac structural defects in embryos lacking Sirt1 in the SHF with and without CoCl2 treatment. Summary of cardiac structural defects in wildtype (Sirt1+/+; Sirt1fl/+/Isl1-Creneg), heterozygous (Sirt1fl/-/Isl1-Creneg; Sirt1fl/+/Isl1-Crepos) and homozygous (Sirt1-/-; Sirt1fl/-/Isl1-Crepos) Sirt1 mutant embryos detected by H&E staining of paraffin sections from E15.5 mouse hearts without (11 embryos from 3 litters mated for germline null embryos and 13 embryos from 4 litters mated for conditional null embryos) or with (27 embryos from 4 litters) CoCl2 treatment. The incidence of different cardiac defects in CoCl2 or untreated hearts with CHDs is indicated. RV: right ventricle; VSD: ventricular septal defect; ASD: atrial septal defect; OA: overriding aorta.



Suppl. Table 3: List of primers used for genotyping, chromatinIP and mutagenesis in the study

Name Primer sequence (5'-->3') Application mSirt1loxP P3 F GGCAGTATGTGGCAGATT Floxed and deleted Sirt1 genotyping mSirt1loxP P4 R CCTGAAACAGACAAGACCT Floxed Sirt1 genotyping mSirt1loxP P6 R GAACATAACAGCCAGGCAT Deleted Sirt1 genotyping Isl1F ACTATTTGCCACCTAGCCACAGCA Isl1-Cre genotyping Isl1R AATTCACACCAAACATGCAAGCTG Isl1-Cre genotyping CreR CTAGAGCCTGTTTTGCACGTTC Isl1-Cre genotyping Nkx2.5F TAAACTGGTCGAGCGATGGATTTCC Nkx2.5-Cre genotyping Nkx2.5R CATATCTCGCGCGGCTCCGACACGG Nkx2.5-Cre genotyping nGFPF CTCTTGATTCCCACTTTGTGGTTC Isl1-nGFP genotyping nGFPR TCAGTAAGCTATGGGTTAGAG Isl1-nGFP genotyping Isl1ISHF GGTCCCGAGCCGTGCAGGTCC DNA probe for ISH Isl1ISHR GCGCGCTGGATGCAAGGGACTG DNA probe for ISH Hes1mF GGACCTACCGTCGACCTACTCGCCACGGGCGGCAG Mutagenesis Hes1mR GGCGAGTAGGTCGACGGTAGGTCCTTCCTGTG Mutagenesis Isl1P-KpnI GGTACCTTGGAGAGCTCAGATTGG Luciferase reporter Isl1P-HindIII AAGCTTATCTGTAAGAGGGAGTAATG Luciferase reporter HA-Nkx2.5 CTAGCTAGCACGATGTACCCATACGATGTTCCAGAT Nkx2.5 expression vector TACGCTTTCCCCAGCCCTGCGCTC

Nkx2.5-V5 ACGCGTCGACCTACGTAGAGTCGAGACCGAGGAGA Nkx2.5 expression vector GGGTTAGGGATAGGCTTACCCCAGGCTCGGATGCCG Isl1p-2940 GCGCCAGGAACTGTGCTCCAA ChIP AGGGGCGACCTCTTGTGTTCAATG Isl1p-850 GAACAGGAGACCTCACGGGTCGGG ChIP CTAGCAGCGCGCTACGCGTTAGGG Nkx2.5p-4604 TTTCTCAACCTTTTCGCCTATTCA ChIP GTTTTCTCCACCCCTTCATCTG Nkx2.5p-9556 GTGCCCCAGTGACCCGCTCCAT ChIP TATCTCCCTTCCCCGCTGTTGTCC Nkx2.5p-2581 AGGCAAAGAAATCACTCCACA ChIP TGTTACAATGGCTGGGAA



Suppl. Table 4: List of primers used for qPCR in the study

Name Primers Application Ta Efficiency Amplicon Position (ºC) (%) size (bp) (start) Isl1 CTGCGGGAGGATGGGCTTTTCT qRT 61 97 179 631 GGTCTTCTCCGGCTGCTTGTGG Flk1 GGGATGGTCCTTGCATCAGAA qRT 58 100 139 4036 ACTGGTAGCCACTGGTCTGGTTG Nkx2.5 ACCTTTCTCCGATCCATCCCACT qRT 58 100 227 1504 GCGTTAGCGCACTCACTTTAATG CD31 GCTCATTGCGGTGGTTGTCAT qRT 58 95 106 2003 CATCTCCACGGGTTTCTGTTTG Myh11 CGCCCAGAAAAACAATGCCCTAAA qRT 61 100 167 3458 GCGTATCCTCCAGCTCCGTCTTGA Myh6 GCCCAGTACCTCCGAAAGTC qRT 61 100 110 239 GCCTTAACATACTCCTCCTTGTC Hand1 AAGACTCTGCGCCTGGCTACCA qRT 61 98 205 761 CGCCCTTTAATCCTCTTCTCGC Hand2 ACTCAGAGCATCAACAGCGCCTTC qRT 61 95 242 1258 TGTGCTTTTCAAGATCTCATTCAGCTC Mef2c GAGCAGTTCTGTGTTCTTTTGC qRT 53 96 129 1 ATCCCTCTGCACAAGTGTCTG Tbx5 ACTGGCCTTAATCCCAAAAC qRT 53 100 210 927 GGTGAGTTTGAGCTT CTGGA Sma TCAGCGCCTCCAGTTCCT qRT 56 100 69 1289 AAAAAAAACCACGAGTAACAAATCAA Myh7 GCCAACACCAACCTGTCCAAGTTC qRT 63 100 179 5806 TGCAAAGGCTCCAGGTCTGAGGGC β-actin CATGAAGATCCTGACCGAGCGTGG qRT 61 95 108 676 TGCTCGAAGTCTAGAGCAACATAGC Sirt1 GCAGGTTGCAGGAATCCAA qRT 55 98 62 1094 GGCAAGATGCTGTTGCAAA 36b4 TCCAGGCTTTGGGCATCA qRT 56 100 74 534 CTTTATCAGCTGCACATCACTCAGA Isl1p-468 AAAGCGGCCCGTTCCAAGTGC ChIP 61* 99 178 -468 GCGCCGCGTCGTGTCCTG Nkx2.5p-9040 AAAGTCCCCGCGAGTGTTGTGT ChIP 61* 100 183 - 9040 TTGGTGAAAAGCGGGATGGAGACG Nkx2.5p-4604 TTTCTCAACCTTTTCGCCTATTCA ChIP 61 93 234 - 4604 GTTTTCTCCACCCCTTCATCTG Nkx2.5p-9556 GTGCCCCAGTGACCCGCTCCAT ChIP 63 100 201 - 9556 TATCTCCCTTCCCCGCTGTTGTCC Nkx2.5p-2581 AGGCAAAGAAATCACTCCACA ChIP 55 93 155 - 2581 TGTTACAATGGCTGGGAA qRT: Real-Time Quantitative Reverse Transcription PCR; *: PCR contains 4% DMSO.



Suppl. Table 5: List of antibodies used in the study.

Antibody Use Use Supplier Cat. No. Histone H3 WB, ChIP Abcam ab1791 H3K9ac WB, ChIP Abcam ab4441 H4K16ac ChIP Abcam ab10158 Isl1 IF, WB, ChIP Hybridoma Bank 39.4D5 Nkx2.5 IF, WB Abcam ab35842 Nkx2.5 WB Santa Cruz Sc-376565 V5 WB Abcam Ab9116 HIF1α WB, IP Bethyl A300-286A HIF1α ChIP Novus Biologicals NB100-134 CD31-APC FACS eBioscience 17-0311 cTNT FACS Abcam ab8295 Myh11-PE FACS Santa Cruz Biotechnology sc-6956 Flk1-PE FACS BD Biosciences 555308 Sirt1 IF, WB, IP Cell signaling 2028 Sirt1 ChIP Millipore 07-131 pHistone 3 IF Cell signaling 9701 Hes1 WB, IP Santa Cruz sc-25392 JNK 1/2 WB Cell signaling 9258 pJNK1/2 WB Cell signaling 9251 α-Tubulin WB Sigma T6074 α-sarc.-Actinin WB Sigma A7811 α-Actin WB Sigma A5441 M2(Flag) WB, IP Sigma A2220 Myc WB, IP Cell signaling 2278 HDAC1 WB, ChIP Cell signaling 5356 HDAC5 WB, ChIP Active Motif 40970

IF: Immunofluorescence, WB: Western blot, IP: immunoprecipitation, ChIP: Chromatin